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INTRODUCTION
The Indian subcontinent boasts a rich heritage of traditional medicine, with various health care systems flourishing for centuries. It mainly consists of three major systems of medicine: Ayurveda, Siddha, and Unani (Wadnerwar et al., 2023; Pratap et al., 2026). The medicinal importance of approximately 20,000 plants has been documented in India, but traditional practitioners use only 7,000-7,500 of them to treat various diseases. Every traditional system has its own flora: Ayurveda about 2500 plants, Siddha about 1350 plants, Unani about 350, Homoeopathy about 850, Tibetan about 550, Modern about 250, and folk about 4500 (Hussain & Hussain, 2010). In every system of medicine, quality control has been considered a significant task to verify the authenticity of drugs from the time of inspection onward, to prevent adulteration and its harmful effects. The plants from Ayurveda, Unani, and Siddha have their own pharmacopoeias with standardization methods. But plants of ethnobotanical importance lack standardized methods for authenticating drugs (Pratap et al., 2026). In such cases, pharmacognostical standardization is needed to authenticate the drug. With this view, a study was undertaken to produce a record documenting the microscopical, macroscopical, phytochemical and HPTLC profile of Elaeodendron glaucum (Rottb.) Pers. is an ethno-medicinally important plant (Pratap et al., 2021). Elaeodendron glaucum is known for its sternutatory action and is employed to relieve severe headache and skin infections (Malpani et al., 2019). It is also very effective in controlling hysteria. The bark is employed to treat Dysuria, wounds, burning sensations, and hyperacidity (Johnson et al., 2015), and the leaves are an effective remedy for cough with phlegm, swellings, and menstrual disorders (Bharath & Suryanarayana, 2008; Jagtap et al., 2009). There has already been significant research on the pharmacological and ethnobotanical properties of Elaeodendron glaucum, but identifying it in dried form from other Elaeodendron species remains a challenge. A review of the existing literature on this plant highlights a lack of standardized studies. Therefore, there is an urgent need for such standardization. To fill this gap, the present study was conducted (Pratap et al., 2012).
MATERIALS AND METHODS
The plant material was collected from Yenugu Mallamma konda, Annamayya district, Andhra Pradesh, in August 2022. The collected specimen was authenticated by Gudivada Sudharshanam, Professor of Botany, using local floras, and the specimen was deposited in the herbarium of the Department of Botany, Sri Venkateswara University, and assigned herbarium reference no. SVUH-1271 for future reference (Gamble, 2005; Jain & Rao, 1976; Pratap et al., 2009, 2010; Pullaiah, 2018; Sekhar et al., 2011).
Evaluation of Macroscopic and organoleptic characters
The collected raw drugs were observed with the naked eye, and their macroscopic characters were noted. The organoleptic characteristics were documented by using standard procedures as follows (Jyothi et al., 2015, 2020; Kumari et al., 2019, 2021,2020,2022; Pratap et al., 2012, 2016,2021,2024,2026; World Health Organization, 2000).
Evaluation of Microscopic characters
The crude or fresh material of the leaf and stem bark was subjected to free-hand sections, and microscopic observations were performed using standard procedures as follows (Jyothi et al., 2015, 2020; Kumari et al., 2019, 2021,2020,2022; Pratap et al., 2012, 2016,2021,2024,2026; World Health Organization, 2000).
Evaluation of powder microscopic characters
The leaf and stem bark powders were treated with different reagents and subjected to microscopical observations using following standard procedures (Jyothi et al., 2015, 2020; Kumari et al., 2019, 2021,2020,2022; Pratap et al., 2012, 2016,2021,2024, 2026, World Health Organization, 2000).
Physicochemical analysis
The plant material was subjected to various physicochemical parameters according to standard procedures (Anonymous, 1996; Evans, 2002; Jyothi et al., 2015, 2020, 2024; Khandelwal, 2008; Kumari et al., 2019, 2020,2021,2022; Pratap et al., 2012, 2016,2021,2024,2026; WHO., 2000).
Phytochemical investigation
The leaf and stem bark powders were extracted with water, methanol, chloroform, ethanol, and hexane. The extracts were subjected to preliminary phytochemical screening according to standard procedures, as follows (Brain & Turner, 1975; Jyothi et al., 2015, 2020; Khandelwal, 2008; Kumari et al., 2019, 2021,2022; Pratap et al., 2012, 2014,2016,2021,2024,2026; WHO., 2000).
Fluorescence analysis
The powdered plant material was subjected to different chemical reagents, and the resulting colour reactions were observed under visible as well as Ultraviolet (UV) light (Anonymous, 1996; Evans, 2002; Kumari et al., 2019, 2021; Pratap et al., 2012, 2020, 2021,2024, 2026; WHO., 2000).
High-Pressure Thin-Layer Chromatography of Leaf in Ethanol Extract
The leaf extract was filtered and concentrated to approximately 5 mL, after which it was analyzed using Thin-Layer Chromatography (TLC). About 7 µL of the ethanol extract was carefully applied onto the TLC plate. The plate was developed using a mobile phase consisting of toluene, ethyl acetate, and methanol in the ratio of 7:2:1 (v/v/v).
After chromatographic development, the plate was air-dried and observed under UV light at 254 nm and 366 nm. Subsequently, the plate was sprayed with vanillin–sulphuric acid reagent and heated at 110°C for approximately 5 min. The developed plate was then examined under visible light. Several distinct spots were detected on the chromatogram, and the plate was photographed for documentation as shown in Figure 1.
HPTLC fingerprint of the Stem bark in ethanol extract
The applied methodology was the same as HPTLC of leaf, but the mobile phase was changed to Chloroform: Methanol (6.5:2, v/v) (Kumari et al., 2020, 2022; Pratap et al., 2021, 2024,2026).
RESULTS
Taxonomy of the plant (Macroscopical characters)
Elaeodendron glaucum is a small tree species. The leaves are sub-opposite, with margins that are entire or slightly crenate. The petiole is up to 2 cm long. The leaf blade is ovate to ovate-elliptic, somewhat coriaceous, with crenate margins, measuring up to 12 × 6.5 cm. The cymes are dichotomously branched and generally shorter than the leaves. Pedicels are about 0.2 cm long. The petals are white and glabrous, while the anthers are yellow. The disc is green and obovoid. The fruit is a drupe, less than 1 cm long, usually containing a single seed (Table 1).
| Sl. No. | Macroscopic Parameter | Leaf | Stem bark |
|---|---|---|---|
| 1. | Appearance | Simple, coriaceous, glabrous, shriveled, petiolate, shows crenate-serrate margin, reticulate venation prominent on lower surface. | Dried rectangular thick pieces of varying sizes, with horizontal and transverse striations, transversely fissured with vertical cracks and exfoliating in irregular scales numerous lenticels and constrictions present. |
| 2. | Size & shape | 8-10 cm long and 3-4 cm width in ovate-oblong, apex acute-acuminate, Petiole 1.5-2 cm, woody. | Up to 1.5 cm in thick, irregularly rectangular pieces. |
| 3. | Colour | Upper light green, lower surface greyish green. | External surface reddish-brown colour, Internal surface cream coloured. |
| 4. | Texture | Smooth. | Rough with longitudinal striations, fracture short. |
| 5. | Oduor | Characteristic. | Characteristic. |
| 6. | Taste | Prominently bitter, slightly astringent. | A Stringent and bitter. |
Organoleptic properties
They were assessed by the sensory organs. The results were depicted in the Table 1.
Microscopy
Leaf
Midrib
TS of midrib shows a single layer of upper and lower epidermal cells with thick cuticle; both epidermis embedded with prismatic and cluster of calcium oxalate, upper epidermis followed by single layer of hypodermis, a patch of collenchymatous tissue below the hypodermis present in the central region, but continuous as a band above the lower epidermis, embedded with brownish content; centrally located one well developed large vascular bundles in shape of are beneath the two smaller vascular bundles, upper two vascular bundles surrounded by continuous ring of sclerenchymatous fibres, while the lower vascular bundles is surrounded by discontinuous ring of sclerenchymatous fibres, phloem containing cluster crystal of calcium oxalate (Figures 1-3).
Lamina
Dorsiventral, single layer of upper and lower epidermal cells with thick cuticle, a single layer of hypodermis present just below the upper epidermis; two rows of palisade cells beneath the hypodermis and spongy parenchyma; both epidermis embedded with calcium oxalate-prismatic cluster crystals; mesophyll tissue embedded with vascular strands (Figures 4 and 5).
Petiole
Transfer section of petiole is irregularly circular in outline, with a single layer of epidermis with very thin cuticle, several layers of parenchymatous tissue beneath the epidermis; parenchymatous tissue of the cortical region contains cluster and prismatic calcium oxalate crystals. Bicollateral vascular bundles are centrally located and capped fully by a ring of grouped pericyclic fibres. In the centre of the petiole, parenchymatous pith is noticed with intercellular spaces (Figure 6).
Powder microscopy of Leaf
The fragments of upper and lower epidermis are arranged with straight anticlinal walls. Anomocytic stomata present on lower epidermis in surface view; both epidermis embedded with cluster, rosette and prismatic crystals of calcium oxalate. There are fragments of fibres with thick and thin lumen, forked, pitted, sharp and blunt ends. Vessels exhibit spiral thickenings and few parenchyma with crystals embedded, a a few parenchyma cells contain embedded crystals. Additionally, some parenchymatous cells display reddish-brown contents. The starch grains are few and consist of a single component, measuring up to 30 µm. The stomatal index ranges from 9.99 to 12.46 on the lower surface, with a stomatal number between 141 and 187 (Figure 7).
Stem bark
Detailed TS of stem bark shows a wide zone of cork and cortex, forming a rhytidoma, cork consist of tangentially elongated rectangular thin-walled cells with reddish brown content, group of stone cells in patches in outer and inner cortex, inner cortex consists of thin-walled parenchymatous cells embedded with group of stone cells and reddish content: followed by wider zone of phloem tissue (Figures 8 and 9).
Powder microscopy of the stem bark revealed characteristic pharmacognostic features including polygonal cork cells, cork cells with embedded stone cells, blunt-ended fibres, thick-walled and pitted stone cells, stout sclereids, simple and compound starch grains, and prismatic calcium oxalate crystals under polarized light (Figure 10A-10H). These diagnostic microscopic characters are valuable for the identification, authentication, and quality control of the stem bark .
Physicochemical
The results for the physicochemical analysis, fluorescence analysis, and preliminary phytochemical screening for both leaf and stem bark are summarized in Tables 2-4, respectively
| Sl. No. | Parameters | Leaf (%) w/w | Stem bark (%) w/w |
|---|---|---|---|
| 1. | Loss on Drying | 0.88 | 9.17 |
| 2. | Total ash | 8.34 | 3.25 |
| 3. | Acid insoluble ash | 0.86 | 0.19 |
| 4. | Water soluble extractive value | 39.33 | 14.32 |
| 5. | Alcohol soluble extractive value | 20.35 | 16.65 |
| 6. | Ph (10 % aqueous solution) | 6.52 | 6.3 |
| Sl. No. | Treatment | Leaf | Stem bark | ||
|---|---|---|---|---|---|
| Visible light | UV light | Visible light | UV light | ||
| 1. | Powder as such | Green | Green | Reddish brown | Brown |
| 2. | Powder+50% HNO3 | Yellow | Green | Yellowish brown | Light brown |
| 3. | Powder+ 1 N HCl | Pale yellow | Yellowish green | Reddish brown | Reddish brown |
| 4. | Powder+50% H2SO4 | Yellowish green | Green | Reddish brown | Reddish brown |
| 5. | Powder+5% Ferric chloride | Green | Green | Deep brown | Black |
| 6. | Powder+ H2O | Green | Green | Brown | Brown |
| Sl. No. | Phytoconstituents | Chemical tests | Plant Part | Water extract | Methanol extract | Chloroform extract | Ethanol extract | Hexane extract |
|---|---|---|---|---|---|---|---|---|
| 1. | Alkaloids | Dragendorff’s test | Leaf Stem bark | -- -- | ++ ++ | ++ ++ | ++ ++ | ++ ++ |
| Mayer’s test | Leaf Stem bark | -- ++ | ++ ++ | ++ ++ | ++ ++ | ++ ++ | ||
| 2. | Glycosides | Borntrager’s test | Leaf Stem bark | ++ ++ | ++ ++ | -- -- | ++ ++ | -- -- |
| 3. | Flavonoids | Test with Mg ribbon and Conc. HCl | Leaf Stem bark | -- ++ | ++ ++ | ++ -- | ++ ++ | -- -- |
| 4. | Protein | Millon’s test | Leaf Stem bark | ++ ++ | -- -- | -- -- | -- -- | -- -- |
| 5. | Tannins | Test with Ferric chloride | Leaf Stem bark | ++ ++ | ++ ++ | -- -- | ++ ++ | -- -- |
| 6. | Phenols | Test with FeCl3 | Leaf Stem bark | -- ++ | ++ ++ | ++ ++ | ++ ++ | -- -- |
| 7. | Carbohydrates | Molisch’s test | Leaf Stem bark | ++ -- | -- -- | -- -- | -- -- | -- -- |
| 8. | Reducing Sugars | Fehling's test | Leaf Stem bark | ++ ++ | ++ -- | -- -- | -- -- | -- -- |
| 9. | Saponins | Froth test | Leaf Stem bark | ++ ++ | ++ ++ | -- -- | + + ++ | -- -- |
HPTLC Screening
Retention factor (Rf) values and corresponding spot characteristics were recorded and are presented in the respective tables (Tables 5-10). Densitometric chromatograms (densitogram) were generated to support qualitative and quantitative evaluation of the phytochemical profile. For the leaf ethanol extract, HPTLC analysis revealed the presence of multiple phytoconstituents (Figure 11). Under wavelength of λmax 366 nm UV light, nine distinct bands were observed with Rf values of 0.01, 0.09, 0.15, 0.25, 0.31, 0.53, 0.81, 0.93, and 0.98, indicating the presence of several fluorescent compounds (Figure 12). The bands exhibited variable fluorescence intensity, suggesting differences in concentration of the constituents. At wavelength of λmax 254 nm, four spots were detected at Rf values 0.01, 0.09, 0.31, and 0.98, (Figure 13) appearing as light-absorbing zones, confirming the presence of UV-active compounds. After derivatization with vanillin–sulphuric acid reagent and scanning at wavelength of λmax 576 nm, five coloured spots appeared at Rf values 0.01, 0.10, 0.28, 0.72, and 0.81, indicating the presence of terpenoids, phenolics, or related secondary metabolites (Figures 14 and 15). The densitogram showed major peaks at Rf 0.01 and 0.10, confirming them as dominant constituents in the leaf extract HPTLC profiling of the stem bark (Figure 16) with ethanol extract, demonstrated a comparatively distinct chemical pattern. When scanned at wavelength of λmax 366 nm, four major fluorescent spots were observed at Rf values 0.004, 0.280, 0.509, and 0.941 (Figure 17). Under wavelength of λmax 254 nm, four absorption bands were detected at Rf 0.01, 0.31, 0.58, and 0.95, (Figure 18) indicating the presence of UV-absorbing phytochemicals. Following derivatization and scanning at wavelength of λmax 576 nm, six well-resolved coloured spots appeared at Rf values 0.01, 0.16, 0.23, 0.34, 0.57, and 0.93, suggesting the presence of diverse classes of secondary metabolites (Figures 19 and 20). The densitometric scan revealed major peaks at Rf 0.57 and 0.93, representing predominant compounds in the stem bark extract. Overall, the HPTLC fingerprint profiles of both leaf and stem bark ethanol extracts demonstrated chemical complexity and distinct phytochemical screenings, which can serve as a reliable reference for identification, quality control, and standardization of the plant material.
| Sl. No. | Max 𝑅f | Max pos. (mm) | Height | Height % | Area | Area % |
|---|---|---|---|---|---|---|
| 1. | 0.01 | 10.40 | 0.33 | 72.90 | 0.0028 | 41.93 |
| 2. | 0.09 | 16.50 | 0.03 | 5.99 | 0.0005 | 7.29 |
| 3. | 0.31 | 31.80 | 0.02 | 5.49 | 0.0008 | 11.62 |
| 4. | 0.98 | 78.60 | 0.07 | 15.63 | 0.0026 | 39.16 |
| Sl. No. | Max Rf | Max pos. (mm) | Height | Height % | Area | Area % |
|---|---|---|---|---|---|---|
| 1. | 0.01 | 10.40 | 0.36 | 43.33 | 0.0028 | 17.01 |
| 2. | 0.09 | 16.40 | 0.01 | 1.65 | 0.0003 | 1.74 |
| 3. | 0.15 | 20.30 | 0.07 | 8.96 | 0.0015 | 9.24 |
| 4. | 0.25 | 27.40 | 0.04 | 4.30 | 0.0008 | 5.06 |
| 5. | 0.31 | 31.90 | 0.09 | 10.90 | 0.0026 | 15.37 |
| 6. | 0.53 | 47.10 | 0.03 | 3.29 | 0.0010 | 5.97 |
| 7. | 0.81 | 66.50 | 0.08 | 9.42 | 0.0033 | 19.69 |
| 8. | 0.93 | 74.80 | 0.09 | 10.64 | 0.0029 | 17.14 |
| 9. | 0.98 | 78.60 | 0.06 | 7.52 | 0.0015 | 8.77 |
| Sl. No. | Max Rf | Max pos. (mm) | Height | Height % | Area | Area % |
|---|---|---|---|---|---|---|
| 1. | 0.01 | 10.70 | 0.46 | 55.29 | 0.0057 | 35.70 |
| 2. | 0.10 | 16.90 | 0.25 | 29.49 | 0.0049 | 30.62 |
| 3. | 0.28 | 29.60 | 0.06 | 6.64 | 0.0023 | 14.26 |
| 4. | 0.72 | 60.10 | 0.03 | 3.55 | 0.0012 | 7.23 |
| 5. | 0.81 | 66.90 | 0.04 | 5.03 | 0.0019 | 12.18 |
| Sl. No. | Max Rf | Max pos. (mm) | Height | Height % | Area | Area % |
|---|---|---|---|---|---|---|
| 1. | 0.01 | 10.7 | 0.2 | 43.9 | 0.0034 | 17.0 |
| 2. | 0.31 | 31.9 | 0.1 | 17.4 | 0.0038 | 18.7 |
| 3. | 0.58 | 50.3 | 0.1 | 13.8 | 0.0034 | 16.8 |
| 4. | 0.95 | 76.2 | 0.1 | 25.0 | 0.0096 | 47.5 |
| Sl. No. | Max Rf | Max pos. (mm) | Height | Height % | Area | Area % |
|---|---|---|---|---|---|---|
| 1. | 0.004 | 10.30 | 0.25 | 63.30 | 0.0016 | 21.17 |
| 2. | 0.280 | 29.60 | 0.04 | 10.37 | 0.0016 | 20.34 |
| 3. | 0.509 | 45.60 | 0.03 | 6.88 | 0.0013 | 16.74 |
| 4. | 0.941 | 75.90 | 0.08 | 19.45 | 0.0032 | 41.75 |
| Sl. No. | Max Rf | Max pos. (mm) | Height | Height % | Area | Area % |
|---|---|---|---|---|---|---|
| 1. | 0.01 | 10.60 | 0.10 | 8.74 | 0.0019 | 3.05 |
| 2. | 0.16 | 21.00 | 0.07 | 6.70 | 0.0018 | 2.91 |
| 3. | 0.23 | 26.40 | 0.03 | 3.07 | 0.0009 | 1.39 |
| 4. | 0.34 | 33.70 | 0.28 | 24.90 | 0.0120 | 19.33 |
| 5. | 0.57 | 50.10 | 0.38 | 34.54 | 0.0251 | 40.31 |
| 6. | 0.93 | 75.30 | 0.24 | 22.05 | 0.0206 | 33.02 |
DISCUSSION
The present study aimed to establish pharmacognostic standards for the leaf and stem bark of Elaeodendron glaucum, an important ethnomedical plant. Herbal drugs are often collected from natural habitats and used in their crude form, making proper identification and standardization essential to ensure their quality, safety, and therapeutic value. Macroscopic evaluation of the leaf and stem bark revealed distinct taxonomic characteristics, including morphology, colour, texture, taste, and structural markings. These external features are crucial for the preliminary identification of the crude drug and can help distinguish it from substitutes or adulterants, as well as from related species. Microscopic examination identified key diagnostic features, including a dorsiventral lamina, well-developed vascular bundles, sclerenchymatous fibres, stone cells, and calcium oxalate crystals in various forms. Additionally, powder microscopy revealed characteristic elements, including fibres, vessels, stone cells, crystals, starch grains, and epidermal fragments. These micro-morphological features of the powdered material serve as the basis for identifying the drug in powdered form. Physicochemical parameters, including ash content, loss on drying, extractive values, and pH, were assessed to evaluate the purity and quality of the plant material. A low moisture content indicates a reduced risk of microbial growth and a prolonged shelf life. The ash values provide information about inorganic matter and potential contamination, while extractive values indicate the nature and quantity of active constituents soluble in different solvents. Higher extractive values in polar solvents suggest the presence of polar phytoconstituents. Fluorescence analysis showed characteristic colour changes in the powdered drug under visible and ultraviolet light after treatment with various reagents. This technique is a quick and reliable method for detecting adulteration and confirming the identity of the crude drug. Preliminary phytochemical screening revealed the presence of several bioactive compounds, including in both leaf and stem bark extracts. HPTLC fingerprint profiles of leaf and stem bark showed well-developed bands and various Rf values under 254 nm and 366 nm in fluorescence mode of UV light. The differences in chromatographic patterns between the leaf and stem bark indicate variations in chemical composition. The obtained HPTLC data can serve as a perfect chemical profiling tool for quality control and authentication.
CONCLUSION
This study provides a detailed pharmacognostical standard for the identification and authentication of Elaeodendron glaucum bark and leaves. These plant parts carry a significant ethnomedical value, and their identification in dried form is challenging. The findings from this research will serve as a valuable resource for future studies, including phytochemical analyses, formulation development, and quality assurance of herbal medicines derived from this plant.